Peptide alarmone signalling triggers an auto-active bacteriocin necessary for genetic competence

The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans . We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the 'death effector'. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm.     

Natural genetic transformation of Streptococcus mutans growing in biofilms

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.     

SMU.152 acts as an immunity protein for mutacin IV

Streptococcus mutans, a principal causative agent of dental caries, secretes antimicrobial peptides known as mutacins to suppress the growth of competing species to establish a successful colonization. S. mutans UA159, a sequenced strain, produces at least two major mutacins, mutacins IV and V. Mutacin IV is a two-peptide mutacin encoded by nlmAB genes, which are mapped just upstream of a putative immunity-encoding gene SMU.152. Here we explored the function of SMU.152 as an immunity protein. We observed that overexpression of SMU.152 in two sensitive host strains converted the strains to become immune to mutacin IV. To identify the residues that are important for immunity function, we sequentially deleted residues from the C-terminal region of SMU.152. We observed that deletion of as few as seven amino acids, all of which are highly charged (KRRSKNK), drastically reduced the immunity function of the protein. Furthermore, we identified two other putative immunity proteins, SMU.1909 and SMU.925, which lack the last four charged residues (SKNK) that are present in SMU.152 but contain the KRR residues. Synthetic addition of SKNK residues to either SMU.1909 or SMU.925 to reconstitute the KRRSKNK motif and expressing these constructs in sensitive cells rendered the cells resistant to mutacin IV. We also demonstrated that deletion of Man-PTS system from a sensitive strain made the cells partially resistant to mutacin IV, indicating that the Man-PTS system plays a role in mutacin IV recognition.